Expression and distribution of the transient receptor potential cationic channel ankyrin 1 (TRPA1) in the human seminal vesicles

Abstract Background and Aims The transient receptor potential cationic channel ankyrin 1 (TRPA1), a channel protein permeable to most divalent cations, has been suggested to play a role in mechano‐afferent/efferent signaling (including the release of neurotransmitters) in the human urinary tract (bladder, prostate, and urethra). To date, only a few studies have addressed the expression of this receptor in male and female reproductive tissues. The present study aimed to evaluate human seminal vesicles (SVs) for the expression and localization of TRPA1. Methods SV tissue was obtained from 5 males who had undergone pelvic surgery due to malignancies of the prostate or urinary bladder. The expression of messenger ribonucleic acid (mRNA) specifically encoding for the TRPA1 protein was elucidated by means of reverse transcriptase polymerase chain reaction (RT‐PCR). Using immunohistochemical methods, the distribution of TRPA1 was examined in relation to the endothelial and neuronal nitric oxide synthases (eNOS, nNOS) and the neuropeptides calcitonin gene‐related peptide (CGRP) and vasoactive intestinal polypeptide (VIP). Results RT‐PCR revealed signals related to the expected molecular size of 656 bp. Immunohistochemistry demonstrated that TRPA1 is located in nerves running through the smooth muscle portion of the SV. Here, the protein is in part co‐localized with nNOS and CGRP, whereas no co‐localization with VIP was registered. Dot‐like signals specific for TRPA1 were observed in the cytoplasm of epithelial cells lining the lumen of glandular spaces. The epithelial layer also presented staining for eNOS. The smooth musculature appeared free of immunosignals for TRPA1. Conclusion The results convincingly show the expression of TRPA1 in nerve endings as well as in epithelial cells of the SV. Based on its location in epithelial cells, TRPA1 might be involved in the mechanism of the NO/cyclic guanosine monophosphate (GMP)‐mediated signaling and also the control of secretory function (mediated by cyclic GMP) in the human SV.


| INTRODUCTION
The seminal vesicles (SVs) are important organs involved in the process leading up to seminal emission and ejaculation in males.
There is extensive evidence that the normal function of SV smooth muscle contributes to the facilitation of seminal emission. 1 Hence, ejaculatory dysfunction, including premature ejaculation and anejaculation, has been suggested as the possible result of disturbances on the level of neuromuscular control. 2,3 Nevertheless, in contrast to the knowledge of the pharmacology of the penile erectile tissue, the multiple biochemical signals and interactions mediating the control of the human SV are still poorly understood. To date, several studies have investigated the potential involvement of pathways mediated by alphaadrenoceptors, the serotoninergic system, the cyclic AMP and nitric oxide (NO)/cyclic guanosine monophosphate (GMP) pathways, potassium channels, and Rho-kinase (ROK). 4 can act as a molecular sensor of mechanical stimuli (such as stretch, leading to the activation of Ca 2+ or release of ATP), noxious chemical irritation, and inflammatory processes in the lower urinary tract (urinary bladder, prostate) as well as in male and female genital tissues (corpus cavernosum penis, clitoris, labia minora, vagina). [10][11][12][13] Since disruptions to sensory pathways that underlie the neuronal control may play a crucial role in the pathophysiology of dysfunctions of lower urinary tract organs, the channel has been proposed as a potential drug target for the symptomatic and/or curative treatment of urinary symptoms (e.g., lower urinary tract symptoms [LUTS] secondary to benign prostatic hyperplasia, overactive bladder [OAB]/detrusor overactivity) and sexual dysfunctions in both sexes. With regard to the ejaculatory response, an association has been postulated between the activity of TRP channels located in the epithelial layer of the glans penis, acting as physiological receptors of physical sensations such as movement of the penis and vaginal humidity, with the condition of lifelong premature ejaculation. 14 Since, to the best of our knowledge, no study has yet investigated TRPA1 in SVs, our study aimed to determine by means of molecular biology

| Immunohistochemistry
Following immersion-fixation for 4 h in 4% formaldehyde in phosphate-buffered saline (pH 7.4), tissues were rinsed several times with phosphate-buffered saline containing 15% (w/w) sucrose and then embedded in Tissue-Tec (Miles Laboratories). Using a cryostat, tissue specimens were sliced to 8-10 µm thickness and thawmounted onto glass slides. Preincubation of sections was done for 2 h in phosphate-buffered saline with 0.2% Triton X-100 and 0.1% bovine serum albumin. The sections were then exposed to the primary antibodies (ABs) directed against TRPA1 (working dilution 1:500), calcitonin gene-related peptide (CGRP, 1:500), neuronal nitric oxide synthase (nNOS, 1:1.000), endothelial NOS (eNOS) and vasoactive intestinal polypeptide (VIP) for 24 h. The sections were rinsed and incubated for 90 min with Alexa Fluor-conjugated secondary ABs (1:800). Thereafter, sections were mounted in phenylendiamine, and visualization was done using a laser fluorescence microscope (Olympus Corp.). 12 Images were created using the ViewFinder program (Pixera). Negative controls without the primary ABs were also performed.   is implicated in the physiology and/or pathophysiology of the cardiovascular, respiratory and gastrointestinal systems and the urogenital tract. The channel protein is permeable to Ca 2+ , acts as a chemo-, mechano-and also thermosensor, and is also involved in the local release of neuropeptides and prostanoids derived from the cyclooxygenase pathway. In addition, the role of TRPA1 in the physiological response to noxious provocation and the involvement of the ion channel in pain and inflammation emphasizes its importance in medicine. Besides being reactive to temperature, it also interacts with endogenous signaling molecules, natural compounds from plants (e.g., mustard, wasabi, garlic, horseradish, or cinnamon), and other chemicals (e.g., paraben, acrolein). [15][16][17] In the human urogenital tract, where NO is an established signaling molecule, modulatory functions by NO on the activity of TRPA1 have been suggested. Furthermore, dihydrogen sulfide (H 2 S), another endogenous signaling molecule that activates TRPA1, has been reported to be involved in the regulation of functions of the lower urinary tract and cardiovascular system and the mediation of neurogenic inflammation but is also produced by invading uropathogens. [18][19][20] TRP channels, in particular TRPV1, TRPV2, TRPV4, TRPM8 and TRPA1, have emerged as key regulators of sensory processes in the lower urinary tract and have thus been investigated as potential targets to treat genitourinary diseases, such as the OAB, LUTS, interstitial cystitis associated with bladder disorder, chronic pelvic pain syndrome and vulvodynia. [21][22][23] The increasing importance of TRP channels in research in the field of urological pharmacology has prompted us to investigate the expression and distribution of TRPA1 in human SV. Our findings demonstrate that the TRPA1 protein is mainly present in slender nerve fibers transversing the smooth muscle portion of the SV. These fibers are seen localized in close relation to varicose nerves containing nNOS and, to a lesser degree, the neuropeptide CGRP. In fact, NO has been reported to be involved in the regulation of tension in isolated SV smooth muscle. 8 As such, it is tempting to speculate that signals mediated via TRPA1 may be linked to the control of smooth muscle tension. This has previously been described for other parts of the urogenital tract.

| AB source
Tissue bath studies have demonstrated that the contraction of the isolated smooth muscle of the human ureter, urethra and prostate, mediated either by alpha-adrenergic agonists or transmural electrical stimulation, was significantly attenuated by stinging natural compounds (allyl isothiocyanate, diallyl disulfide, cinnamonaldehyde) and sodium hydrogen sulfide (NaHS), known to bind to TRPA1 and activate the channel to act as pain-/mechanosensor at the cellular level. 10,17,24,25 No such functional experiments have yet been conducted using animal or human SV. Some, but not all CGRPpositive terminals also expressed TRPA1. This may suggest that TRPA1 is expressed in subpopulations of sensory nerves. Thus, one can propose that TRPA1 may act as a detector of wall stretch and/or a sensor of the chemical composition of the SV tissue. The latter theory also relates to our finding that TRPA1 nerve varicosities extend into the epithelium. We also located TRPA1 in epithelial cells of glandular ducts that also stained for the endothelial isoform of the NOS (eNOS). NO has previously been related to the secretory functions of the SV. 26 The location of TRPA1 in the epithelium forms a morphological basis for the involvement in secretory function.
Interestingly, cinnamonaldehyde has been shown to alter both the flow rate and composition of salivary gland secretions in human volunteers. 27 Up until today, TRPA1 has been investigated in other male and female genital tissues: In the human clitoris and vagina, TRPA1 was shown to be expressed in slender varicose nerve fibers transversing subepithelial layers. These nerves were also immunoreactive for nNOS and, in the vaginal wall, the CGRP. In clitoral epithelial cells, TRPA1 was also found co-localized with vimentin, known as a specific feature of interstitial/neuroendocrine cells. In the human corpus cavernosum, immunoreactivity for TRPA1 was seen in nerves transversing the cavernous sinusoidal space and running alongside small penile arteries. Here, in contrast to the observations in human SV, these varicose nerves did not stain for nNOS but displayed expression of the vesicular acetylcholine transporter protein. The patterns seen in the clitoris and vagina suggest an involvement of the TRPA1 receptor in both afferent and efferent transmission, that is, in part, connected to the NO/cyclic GMP pathway. 12,13 A link between TRPA1 and (sensory) neurotransmission has also emerged from studies on tissues of the upper and lower urinary tract (ureter, urinary bladder, prostate, urethra). Tissue bath studies demonstrated that the contraction of the isolated smooth muscle of the human ureter, urethra and prostate, mediated either by alpha-adrenergic agonists or transmural electrical stimulation, was significantly attenuated by stinging natural compounds (allyl isothiocyanate, diallyl disulfide, cinnamonaldehyde) and NaHS, known to bind to TRPA1 and activate the channel to act as pain-/mechanosensor at the cellular level. 11,18,24 In contrast, no such functional experiments have yet been conducted using animal or human SV.
In conclusion, the current study detected TRPA1 at both the transcriptional and protein level in the human SV. TRPA1 was detected at both the transcriptional and protein level in the human SV. As signals mediated by NO previously have been linked to secretory functions of the SV, one can speculate that the localization of the cationic channel in slender nerves and epithelial cells epithelium of glandular ducts form a basis for a possible role for TRPA1 in modifying secretory function. This and whether or not TRPA1 is also involved in mechano-afferent signals and/or can modulate SV smooth muscle function by acting as an entry gate for Ca 2+ into the cytosolic space needs to be further explored in functional settings. had full access to all data from this study, and take complete responsibility for the integrity of the data and accuracy of data analysis.

ACKNOWLEDGMENT
Open Access funding enabled and organized by Projekt DEAL.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Original data from the experiments are available from the corresponding author. Previously reported bibliographic data, available at PubMed/Medline, were used to support the results of this study.
These prior studies are cited within the manuscript text and listed in the references.

TRANSPARENCY STATEMENT
The lead author Stefan Ückert affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.